A major limitation to the use of microelectrode technologies in both research and clinical applications is our inability to consistently record high quality neural signals. There is increasing evidence that recording instability is linked, in part, to neuroinflammation. A number of factors including extravasated blood products and macrophage released soluble factors are believed to mediate neuroinflammation and the resulting recording instability. However, the roles of other inflammatory stimuli, such as residual endotoxin contamination, are poorly understood. Therefore, to determine the effect of endotoxin contamination we examined the brain tissue response of C57/BL6 mice to non-functional microelectrodes with a range of endotoxin levels. Endotoxin contamination on the sterilized microelectrodes was measured using a limulus amebocyte lysate test following FDA guidelines. Microelectrodes sterilized by autoclave, dry heat, or ethylene oxide gas, resulted in variable levels of residual endotoxins of 0.55 EU/mL, 0.22 EU/mL, and 0.11 EU/mL, respectively. Histological evaluation at two weeks showed a direct correlation between microglia/macrophage activation and endotoxin levels. Interestingly, astrogliosis, neuronal loss, and blood brain barrier dysfunction demonstrated a threshold-dependent response to bacterial endotoxins. However, at sixteen weeks, no histological differences were detected, regardless of initial endotoxin levels. Therefore, our results demonstrate that endotoxin contamination, within the range examined, contributes to initial but not chronic microelectrode associated neuroinflammation. Our results suggest that minimizing residual endotoxins may impact early recording quality. To this end, endotoxins should be considered as a potent stimulant to the neuroinflammatory response to implanted intracortical microelectrodes.
Multi-beam scanning electron microscopy (mSEM) enables high-throughput, nano-resolution imaging of macroscopic tissue samples, providing an unprecedented means for structure-function characterization of biological tissues and their cellular inhabitants, seamlessly across multiple length scales. Here we describe computational methods to reconstruct and navigate a multitude of high-resolution mSEM images of the human hip. We calculated cross-correlation shift vectors between overlapping images and used a mass-spring-damper model for optimal global registration. We utilized the Google Maps API to create an interactive map and provide open access to our reconstructed mSEM datasets to both the public and scientific communities via our website www.mechbio.org. The nano- to macro-scale map reveals the tissue’s biological and material constituents. Living inhabitants of the hip bone (e.g. osteocytes) are visible in their local extracellular matrix milieu (comprising collagen and mineral) and embedded in bone’s structural tissue architecture, i.e. the osteonal structures in which layers of mineralized tissue are organized in lamellae around a central blood vessel. Multi-beam SEM and our presented methodology enable an unprecedented, comprehensive understanding of health and disease from the molecular to organ length scale.
This study combines novel multibeam electron microscopy with a geographical information system approach to create a first, seamless, navigable anatomic map of the human hip and its cellular inhabitants. Using spatial information acquired by localizing relevant map landmarks (e.g. cells, blood vessels), network modeling will enable disease epidemiology studies in populations of cells inhabiting tissues and organs.
Natural materials exhibit smart properties including gradients in biophysical properties that engender higher order functions, as well as stimuli-responsive properties which integrate sensor and/or actuator capacities. Elucidation of mechanisms underpinning such smart material properties (i), and translation of that understanding (ii), represent two of the biggest challenges in emulating natural design paradigms for design and manufacture of disruptive materials, parts, and products. Microscopy Aided Design And ManufacturE (MADAME) stands for a computer-aided additive manufacturing platform that incorporates multidimensional (multi-D) printing and computer-controlled weaving. MADAME enables the creation of composite design motifs emulating e.g., patterns of woven protein fibers as well as gradients in different caliber porosities, mechanical, and molecular properties, found in natural tissues, from the skin on bones (periosteum) to tree bark. Insodoing, MADAME provides a means to manufacture a new genre of smart materials, products and replacement body parts that exhibit advantageous properties both under the influence of as well as harnessing dynamic mechanical loads to activate material properties (mechanoactive properties). This Technical Report introduces the MADAME technology platform and its associated machine-based workflow (pipeline), provides basic technical background of the novel technology and its applications, and discusses advantages and disadvantages of the approach in context of current 3 and 4D printing platforms.
Using either the principle of minimum energy or constant shear stress, a relation can be derived that predicts the diameters of branching vessels at a bifurcation. This relation, known as Murray's Law, has been shown to predict vessel diameters in a variety of cardiovascular systems from adult humans to developing chicks. The goal of this study is to investigate Murray's Law in vessels from mice that are haploinsufficient for the elastin protein (Eln+/−). Elastin is one of the major proteins in the blood vessel wall and is organized in concentric rings, known as lamellae, with smooth muscle cells (SMCs) around the vessel lumen. Eln+/− mice have an increased number of lamellae, as well as smaller, thinner vessels. It is possible that, due to decreased amounts of elastin available for vessel wall remodeling during development and in adulthood, Eln+/− vessels would not follow Murray's Law. We examined vessel bifurcations in six different physiologic regions, including the brain, heart, epidermis, ceocum (or cecum), testes, and intestines, in Eln+/− mice and wild-type (WT) littermates. All vessels were between 40 – 300 μm in diameter. We found that the diameters of both Eln+/− and WT vessels have an average of 13% error from the diameters predicted by Murray's Law, with no significant differences between genotypes or physiologic regions. The data suggest that vessels are optimized to follow Murray's Law, despite limitations on the proteins available for growth and remodeling of the vessel wall.
The urea channel UT-A1 and the water channel aquaporin-2 (AQP2) mediate vasopressin-regulated transport in the renal inner medullary collecting duct (IMCD). To identify the proteins that interact with UT-A1 and AQP2 in native rat IMCD cells, we carried out chemical cross-linking followed by detergent solubilization, immunoprecipitation, and LC-MS/MS analysis of the immunoprecipitated material. The analyses revealed 133 UT-A1-interacting proteins and 139 AQP2-interacting proteins, each identified in multiple replicates. Fifty-three proteins that were present in both the UT-A1 and the AQP2 interactomes can be considered as mediators of housekeeping interactions, likely common to all plasma membrane proteins. Among proteins unique to the UT-A1 list were those involved in posttranslational modifications: phosphorylation (protein kinases Cdc42bpb, Phkb, Camk2d, and Mtor), ubiquitylation/deubiquitylation (Uba1, Usp9x), and neddylation (Nae1 and Uba3). Among the proteins unique to the AQP2 list were several Rab proteins (Rab1a, Rab2a, Rab5b, Rab5c, Rab7a, Rab11a, Rab11b, Rab14, Rab17) involved in membrane trafficking. UT-A1 was found to interact with UT-A3, although quantitative proteomics revealed that most UT-A1 molecules in the cell are not bound to UT-A3. In vitro incubation of UT-A1 peptides with the protein kinases identified in the UT-A1 interactome revealed that all except Mtor were capable of phosphorylating known sites in UT-A1. Overall, the UT-A1 and AQP2 interactomes provide a snapshot of a dynamic process in which UT-A1 and AQP2 are produced in the rough endoplasmic reticulum, processed through the Golgi apparatus, delivered to endosomes that move into and out of the plasma membrane, and are regulated in the plasma membrane.
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